Flurbiprofen-Sepharose chromatography of the prostaglandin synthetase from bovine seminal vesicles

Flurbiprofen-Sepharose and Acetyl-Sepharose have been prepared by coupling dl-2-(2-fluoro-4-biphenylyl)propionic acid [Flurbiprofen] and acetic acid, respectively, to 3-(N-[3-aminopropyl])aminopropyl Sepharose 4B using a water soluble carbodiimide. The arachidonic acid oxygenase activity of solubilized bovine seminal vesicle microsomes is retarded during chromatography on Flurbiprofen-Sepharose but not Acetyl-Sepharose. Thus binding of the oxygenase to Flurbiprofen-Sepharose results from interaction with the immobilized inhibitor. However, the impure oxygenase is either not bound and/or not eluted in a biospecific manner since the abilities of flufenamic acid, R(+) and S(-)-5-cyclohexylindan-1-carnboxylic acid, and R and S-Naproxen to remove the enzyme from Flurbiprofen-Sepharose do not parallel the relative efficacies of these compounds as prostaglandin synthesis inhibitors. Nevertheless, gradient elution of arachidonic acid oxygenase activity from Flurbiprofen-Sepharose with flufenamic acid provides a 15 fold enrichment of the enzyme from solubilized bovine seminal vesicle microsomes in 80% yield indicating that this chromatographic reagent can be a powerful tool for use in purification of the prostaglandin synthetase.     

Properties of solubilized prostaglandin synthetase from sheep vesicular glands.

Prostaglandin synthetase activity associated with the microsomal fraction from sheep vesicular glands has been solubilized by treatment with the non-ionic detergents Tween 20, Lubrol Px and Lubrol Wx. Approx.8 fold purification from microsomes is obtained and over 90% of the activity is recovered in the detergent solubilized fraction. The solubilized synthetase activity is stable at pH 5.0 but is gradually lost at pH 8.0; it is also heat and acid labile. The relative amounts of prostaglandins E₂, D₂ and F_2α formed by the microsomal-bound synthetase and by the solubilized synthetase are similar. Also similar are the pH optima (7.9–8.5) of the two synthetase preparations. The solubilization process appears to yield a fully active enzymatic preparation which could be employed for further purification and characterization of the prostaglandin synthetase complex.     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

Biochemical properties of the prostaglandin/thromboxane synthetase of human blood platelets and comparison with the synthetase of bovine seminal vesicles

The enzyme system which synthesizes prostaglandins and thromboxanes in extracts of washed human platelets has been characterized with respect to kinetic parameters, pH and cofactor dependence, and inhibitor potencies. Arachidonate and dihomo-γ-linolenate were shown to be mutually competitive substrates, thus providing biochemical support for the assumption that both substrates are metabolized by the same cyclooxygenase, although they are ultimately metabolized to different patterns of products. Products of the synthetase of human leucocytes qualitatively resemble those obtained with human platelets. The prostaglandin synthetase of bovine seminal vesicles was studied under similar conditions, and kinetic parameters and inhibitor potencies were compared with those of platelet extracts.     

The heme-binding properties of prostaglandin synthetase from sheep vesicular gland

Purified, apoprostaglandin synthetase was prepared from sheep vesicular gland and studied in terms of its heme-binding properties. The enzyme binds a single heme group per enzyme monomer, Mr = 70,000. When reconstituted with heme, the enzyme has an absorption maximum at 412 nm and an absorption coefficient, epsilon 412 nm, of 120 mM-1 cm-1. The binding of heme to the apoenzyme was accompanied by a proportional increase in enzyme activity up to the point of heme-binding saturation. This reconstituted holoenzyme forms prostaglandin H2 from arachidonate. We conclude that prostaglandin synthetase possesses the heme-binding properties of a "typical" heme protein and that a single heme group mediates both the oxygenase and the peroxidase activities of the enzyme.     

Observations on the substrate specificity of prostaglandin hydroxylases of monkey seminal vesicles and sheep vesicular glands

Prostaglandin (PG) 19-hydroxylase of monkey seminal vesicles metabolizes PGE1 and PGE2 to their 19-hydroxy metabolites, while PGE2 20-hydroxylase of ram vesicular glands metabolizes PGE2 to 20-hydroxy-PGE2. The purpose of the present study was determine whether PGF2 alpha is a substrate of these enzymes. Deuterated 20-hydroxy-PGF2 alpha was employed as an internal standard to study the hydroxylation of PGF2 alpha (0.2 mM) by microsomes of monkey (Macaca fascicularis) seminal vesicles in the presence of NADPH, and the biosynthesis was compared with the hydroxylation of PGE2 under identical conditions. 19-Hydroxy-PGF2 alpha was formed at a rate of 3.5% of the formation of 19-hydroxy-PGE2. Microsomes of ram vesicular glands also hydroxylated PGE2 more efficiently than PGF2 alpha, which was converted to both 20-hydroxy-PGF2 alpha and 19-hydroxy-PGF2 alpha at a combined rate of 5% of the biosynthesis of 20-hydroxy-PGE2 under the same conditions. 20-Hydroxy-PGF2 alpha was demonstrated in ram semen, but the concentration was low (0.1 microM) in comparison with 20-hydroxy-PGE2 (24 microM). The two genital PG hydroxylases thus metabolize PGF2 alpha much less efficiently than PGE2. This finding may suggest that 19-hydroxy- and 20-hydroxy-PGF2 alpha in seminal fluids also could be formed by other mechanisms, e.g., from 19-hydroxy- and 20-hydroxy-PGE2 by the PGE 9-keto reductase enzyme.     

Biochemical properties of the prostaglandin/thromboxane synthetase of human blood platelets and comparison with the synthetase of bovine seminal vesicles.

The enzyme system which synthesizes prostaglandins and thromboxanes in extracts of washed human platelets has been characterized with respect to kinetic parameters, pH and cofactor dependence, and inhibitor potencies. Arachidonate and dihomo-gamma-linolenate were shown to be mutually competitive substrates, thus providing biochemical support for the assumption that both substrates are metabolized by the same cyclooxygenase, although they are ultimately metabolized to different patterns of products. Products of the synthetase of human leucocytes qualitatively resemble those obtained with human platelets. The prostaglandin synthetase of bovine seminal vesicles was studied under similar conditions, and kinetic parameters and inhibitor potencies were compared with those of platelet extracts.     

Biochemical characterization of prostaglandin 19-hydroxylase of seminal vesicles

The microsomal fraction of homogenates of seminal vesicles of men and monkeys, Macaca fascicularis, were analyzed for prostaglandin (PG) 19-hydroxylase activity. The microsomes of the monkey seminal vesicles, supplemented with 1 mM NADPH, metabolized 0.2 mM PGE1 to 19-hydroxy-PGE1 at a mean rate of 0.26 nmol/min/mg of protein (with an apparent Km and an apparent Vmax of 40 microM and 0.30 nmol/min/mg of protein, respectively). The enzyme catalyzed the incorporation of atmospheric oxygen into the substrate. Substituting NADH for NADPH reduced the prostaglandin E1 19-hydroxylase activity to 40%. Carbon monoxide and proadifen (SKF 525A) inhibited the enzyme. Prostaglandin E2 (0.2 mM) was metabolized to 19-hydroxyprostaglandin E2 (0.2 nmol/min/mg of protein), but PGE1 was preferred as a substrate. Prostaglandin B1 was metabolized to 18-hydroxy-, 19-hydroxy-, and 20-hydroxyprostaglandin B1 at a combined rate of approximately 25% of prostaglandin E1. 19-Hydroxyprostaglandin B1 was the main product. The microsomes of human seminal vesicles metabolized 0.2 mM PGE2 to 19-hydroxy-PGE2 in the presence of 1 mM NADPH, while carbon monoxide inhibited this reaction. These results suggest that prostaglandin 19-hydroxylase of seminal vesicles might be a cytochrome P-450. The biosynthesis of 19-hydroxyprostaglandin E1 and 19-hydroxyprostaglandin E2 was also studied in vivo in man by analysis of the product/substrate ratios (i.e. 19-hydroxyprostaglandin E1/prostaglandin E1 and 19-hydroxyprostaglandin E2/prostaglandin E2) in a series of consecutive ejaculates, which were obtained during short intervals. There was a 10-fold interindividual difference in these ratios. Although the product/substrate ratios decreased, the 19-hydroxylation of E prostaglandins appeared to be efficient in vivo, which was in contrast to the rather slow biosynthesis in vitro.     

Properties of a partially-purified preparation of the prostaglandin —Forming oxygenase from sheep vesicular gland

The fatty acid oxygenase of sheep vesicular glands was solubilized with Tween-40 and purified 60-fold using ammonium sulfate precipitation and DEAE-cellulose chromatography. Glycerol (50%) stabilized the activity at all stages of purification and allowed long-term storage at −60°. The partially purified enzyme contained less than 0.7 nmoles of iron per mg of protein and less than 0.1 nmole of copper per mg of protein. Although the K_I values for aspirin, BL-2338, flurbiprofen and ibuprofen remained relatively unchanged during purification, the apparent K_I value for inhibition by indomethacin decreased from 120 to 2.7 μm.     

Indomethacin and aspirin inhibition of prostaglandin E2 synthesis by sheep seminal vesicles microsome powder

Sheep seminal vesicles microsome powder was used as a source of prostaglandin synthetase in studies on the nature of inhibition of prostaglandin synthesis by indomethacin and aspirin. Irdomethacin inhibition was found to be highly irreversible, although partial recovery of synthetase activity was obtained after extensive dialysis. A major difference was observed between the effects of aspirin and indomethacin on prostaglandin synthetase activity in seminal vesicles slices. Enzyme activity in microsomes prepared from slices incubated with aspirin was markedly inhibited while the activity in microsomes prepared after incubation with indomethacin was not affected. These results suggest that indomethacin may penetrate intracellularly very slowly, or not at all, and raise a question as to whether the inhibition by indomethacin $\text{in vivo}$ is mediated via direct inhibition of prostaglandin synthesis.     

Purification and characterisation of prostaglandin endoperoxide synthetase from sheep vesicular glands.

The membrane-bound prostaglandin endoperoxide synthetase was purified until homogeneity, starting from sheep vesicular glands. The enzyme was obtained as a complex with Tween-20, containing 0.69 mg detergent per mg protein. No residual phospholipid could be detected. Prostaglandin endoperoxide synthetase appeared to be a glycoprotein, containing mannose and N-acetyl-glucosamine. No haemin or metal atoms were present. A molecular weight of 126 000 was found for the apoprotein by ultracentrifugation in 0.1% Tween solutions. The polypeptide chain without carbohydrate had a molecular weight of 69 000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pure enzyme displays both cyclooxygenase and peroxidase activity, thus converting arachidonic acid into prostaglandin H2. The isolated synthetase requires haemin, which possibly acts as an easily dissociable prosthetic group, and a suitable hydrogen donor to protect the enzyme from peroxide inactivation and which is consumed in stoichiometric amounts to reduce the intermediate hydroperoxy group.     

Metabolism of 3-methylindole by prostaglandin H synthase in ram seminal vesicles

3-Methylindole (3MI) causes a highly tissue- and species-selective lesion of the lung. Metabolic activation of 3MI by the NADPH-dependent mixed function oxidase (MFO) system is the initial event in the lung-specific toxicity. One-electron co-oxidation of 3MI by prostaglandin H synthase (PHS) has been implicated as an alternative mechanism for toxicity in the lung that contains high PHS activity. The objective of this study was to determine if 3MI can be co-oxidized by the arachidonic acid dependent PHS complex. Ram seminal vesicle (RSV) microsomes, which lack MFO activity, were used as a source of PHS. Incubations of RSV microsomes with 3MI, at a concentration as low as 0.01 mM, showed an increase in PHS activity, as indicated by an enhanced rate of oxygen consumption. This effect was arachidonic acid dependent and was inhibited (98%) by indomethacin. Addition of 3MI resulted in a concentration-dependent increase in PHS-catalyzed prostaglandin biosynthesis from [14C]arachidonic acid. PHS-dependent oxidative metabolism of [14C]3MI resulted in a twofold increase in ethyl acetate extracted radiolabelled metabolites. ESR spin-trapping studies demonstrated the presence of a 3MI free radical generated from the metabolism of 3MI by horseradish peroxidase, a model system of PHS hydroperoxidase. The results indicate that 3MI can be co-oxidized by the arachidonic acid-dependent PHS complex. Co-oxidation of 3MI by PHS may play a role in the tissue specificity of 3MI-induced pneumotoxicity.     

PI-specific phospholipase C "alpha" from sheep seminal vesicles is a proteolytic fragment of PI-PLC delta.

Phosphatidylinositide-specific phospholipase C enzymes (PLCs) catalyze the conversion of the phosphoinositides to biologically important signal transducing molecules. These enzymes may be grouped into "families" which share similar structures and modes of regulation. The existence of a structurally distinct family of PLC termed "alpha" has been recently called into question. In the current paper we show by immunoblotting experiments that PLC "alpha" from sheep seminal vesicles is recognized by monoclonal antibodies raised against the delta 1 isoform of bovine brain PLC, and appears to be derived from a higher molecular weight band at 85 kDa. We also show that antibodies raised against PLC alpha efficiently immunoprecipitate the 85-kDa PLC delta 1 isoform from bovine brain and Chinese hamster lung fibroblasts. These data provide strong evidence that the PLC alpha from sheep seminal vesicles is a proteolytic fragment of PLC delta 1. Thus, there is still no conclusive evidence for a separate "alpha" class of PLC.     

Stabilization of prostaglandin synthetase by immobilization of goat seminal microsomes on silica gel-G

Prostaglandin synthetase was immobilized by adsorption of goat vesicular microsomes on silica gel containing CaSO4 (silica gel G). Repeated cycles of enzymatic conversion of arachidonic acid to prostaglandin by the immobilized microsomes increased the product yield by 1.5 fold, in comparison to the same by free microsomal particles. The presence of Ca2+ in silica gel is responsible for this improved yield of prostaglandin as the divalent metal ion stabilized prostaglandin synthetase activity in a remarkable way. Microsomal particles immobilized on solid supports like alumina G and controlled pore glass were not very effective.     

Properties of prostaglandin synthetase of rabbit kidney medulla.

The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.